The status of malaria as one of the top three global infectious disease killers, coupled with the recent rapid spread of drug-resistant Plasmodium falciparum parasites, have made the development of an effective P. falciparum vaccine an urgent priority. However the complexity of the P. falciparum life cycle and the size of its genome present a large number of potential vaccine candidates, making it critical that individual antigens are moved down the malaria vaccine development pipeline only when they meet specific go/no-go criteria. This application will apply such rigorous criteria to two outstanding blood stage candidate vaccine antigens, the closely related merozoite surface proteins PfMSP3 and PfMSP6. The long-term objectives of this proposal are to understand how PfMSP3 and PfMSP6 sequences change over time in a malaria-endemic community and to generate critical correlation of protection data for all sub-domains of both candidate antigens. This work is made possible by our access to unique samples that are collected as part of an ongoing longitudinal cohort study in the Peruvian Amazon, the set-up of which allows us to track individuals from their first P. falciparum infection, with sera and P. falciparum DNA samples available from the time of diagnosis through multiple follow-up visits over the course of several years. In Specific Aim 1 we will establish the temporal dynamics of PfMSP3 and PfMSP6 genetic diversity by following inter- and intra-allelic diversity in these two genes both at a population level, between transmission seasons, and at an individual level, between subsequent infections in the same individual. Specific Aim 2 will use sera from individual infections that have been genotyped in Specific Aim 1 to investigate the relative contribution of specific PfMSP3 and PfMSP6 sub-domains to the development of functional anti-malaria immunity. To achieve this we will both follow antibody levels against each sub-domain of PfMSP3 and PfMSP6, and also use inhibition of invasion and antibody dependent cellular inhibition assays to establish the function of these antibodies in inhibiting P. falciparum parasite growth. Because of the unique combination of a hypoendemic study site and a longitudinal cohort design we will be able to track individuals through several rounds of single isolate infections, and establish both the genotype of each infection and the antibody response to that infection. Combining genotyping and antibody data from the two specific aims will therefore allow us to establish whether antibodies generated against one PfMSP3 or PfMSP6 allele are functionally cross-protective against the other, as well as to establish which sub-domains most closely correlate with protection. The rationale is that by understanding the diversity constraints within which a PfMSP3/PfMSP6 vaccine must operate and the relative contribution of each sub-domain to functional anti-malaria immunity, we will be able to apply rational go/no-go criteria to rule specific sub-domains in or out of the vaccine development process. The overall impact on malaria vaccine design, a question of urgent public health importance, will therefore be significant. Plasmodium falciparum parasites kill more than 1 million people each year and the recent rapid spread of drug resistant parasites have made the development of a P. falciparum vaccine an urgent public health priority. This proposal focuses on two outstanding vaccine candidate antigens, P. falciparum Merozoite Surface Proteins 3 and 6, and will use DNA and sera samples collected from a unique ongoing malaria cohort study in Iquitos, Peru, to establish the relative contribution of distinct sub-domains of these antigens to the development of functional anti-malaria immunity. This data will directly impact vaccine design by allowing us to rule specific sub-domains in or out of the ongoing malaria vaccine development process. [unreadable] [unreadable] [unreadable]